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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
  • B01D15/3804—Affinity chromatography
  • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
  • B01J20/3206—Organic carriers, supports or substrates
  • B01J20/3208—Polymeric carriers, supports or substrates
  • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
  • B01J20/3206—Organic carriers, supports or substrates
  • B01J20/3208—Polymeric carriers, supports or substrates
  • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
  • B01J20/30—Processes for preparing, regenerating, or reactivating
  • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
  • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
  • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
  • B01J20/3244—Non-macromolecular compounds
  • B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
  • B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
  • B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B62/00—Reactive dyes, i.e. dyes which form covalent bonds with the substrates or which polymerise with themselves
  • C09B62/02—Reactive dyes, i.e. dyes which form covalent bonds with the substrates or which polymerise with themselves with the reactive group directly attached to a heterocyclic ring
  • C09B62/04—Reactive dyes, i.e. dyes which form covalent bonds with the substrates or which polymerise with themselves with the reactive group directly attached to a heterocyclic ring to a triazine ring
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6456—Plasminogen activators
  • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase

Definitions

• the invention relates to protein-binding media for affinity chromatography and especially to solid media having high specific protein-binding capacity.
• Affinity chromatography involves separation of proteins by selective adsorption onto and/or elution fron a solid medium.
• the solid m edi um is generally an inert carrier matrix to which is attached a ligand having the capacity to bind under certain conditions the required protein or proteins in preference to others present in the same sample, although in some cases the matrix itself may have such selectivebinding capacity.
• the ligand may be biologically complementary to the protein to he separated, for example antigen and antibody or may he any biologically unrelated molecule which by virtue of the nature and steric relationship of its active groups has the power to bi nd the proteins.
• cyanogen bromide activation has serious disadvantages, especially for industrial use; the material is highly toxic and hence too dangerous to use on an industrial scale; it produces arnido carbonate and carbamate links with the matrix which are unstable and cause gradual loss of column activity in long-term use; and it activates hydroxyl and amine groups on the dye resulting in dye-dye bondingand dyebonding other than through the triazine group so that only a small proportion of the bound dye molecules are available for protein binding and their protein binding properties may differ.
• columns with very high bound dye contents bind protein so fir ⁇ ly that elution is not practicable.
• cyanogen bromide activation cannot be used for immobilisation of dichloro-triazinyl dyes.
• a further class of solid media are prepared by linking mono- chloro-triazinyl dyes to dextran and then to agarose supports.
• the chlorine substituent on the triazine ring is replaced by an -O-dextraa linkage and linkage to the support is entirely via other reactive, especially amine, groups on the dye following cyanogen bromide activation.
• the binding specificity again differs from media wherein the binding is via the triazine ring, and the media suffer all the disadvantages discussed above in respect of cyanogen bromide activation.
• the present invention provides a process for producing a protein-binding solid medium, said process comprising reacting a protein-binding ligand material containing chloro-triasinyl or related groups vith an aqueous suspension of a non-cellulosic matrix containiing free hydroxy or amino groups in the presence of an alkali metal hydroxide at a pH of at least 8 and subsequently washing the resulting solid medium to remove unreacted dye.
• terra "related groups” refers to groups which are recognised in the dye art as analogous, for example chloro-pyrimidinylamino or carbonylamino groups
• the use of an alkali metal hydroxide in this reaction results in very much higher binding of the protein-binding ligand to the matrix than occurs with sodium carbonate or other bases under identical conditions of temperature- time and alkali concentration.
• the present invention is capable of producing media showing tighter elution profiles under similar conditions than the media of the prior art.
• the protein-binding ligand may be any protein-binding material containing a mono or di-chloro triazinyl group or related group but is preferably a so-called "triazinyl dye such as those sold under the Trade Marks "Cibacron” and “Procion”. These are normally triazinyl derivatives of sulphonated anthroquinones, thalocyanines or polyaromatic azo compounds. Such compounds have the structure
• R 1 is a sulphonated group derived from anthroquinone, a substituted anthroquinine, an aromatic azo group or a thalocyanino compound and B is either (a) an organic, normally a sul ated aromatic group, especially a sulphonated phenyl group, or (b) a chlorine atom.
• R 1 may have the structure
• R 3 , R 4 , R 5 each represent a sulphonyl group or a hydrogen atom or an alkyl or amino substituted derivation of such structure.
• R 1 may have the structure
• R 6 , R 7 , R 8 are sulphonyl groups or hydrogen atoms and the point of attachment to the triazinyl ring may be any of points marked "*", or substituted derivatives of such a structure.
• numerous other such compounds are known. When commercial dyes are used it may be necessary to remove wetting agents, for example, by washing with ether or acetone.
• the matrix may be any support commonly used for affinity chroiaatography media, for example polymers and copolymers of agarose, dextrose, dextrans and amides, especially acrylatnide. Glass beads or nylon natrices may also be used. Cellulose and substituted celluloses are generally unsuitable since although they bind large weights of dye, this is poorly accessible to the protein resulting in poor protein binding.
• the matrix is an agarose polymer or copolymer.
• alkali metal hydroxide The optimum concentration of alkali metal hydroxide depends on the structure of the ligand. Thus for monochloro-triazinyl derivatives
• the pH should be at least 9.5 to achieve an optimum coupling. Normally the alkali concentration should be 0.02 to
• the alkali concentration should be about 0.002 to 0.1, preferably 0.005 to 0.01N
• the ligand-matrix coupling reaction may be conducted over a wide range of temperature, within the stability ranges of both reagents, without serious effect on the amount of ligand bound.
• the mono-chloro triazinyl ligands bind only slowly so that at ambient temperatures (15-25oC) as long as to to 60 hours may be required for optimum reaction, and elevated temperatures of 40o to 60oC are preferred since they both speed the reaction and yield a media showing a tighter elution profiles.
• Dichloro-triazinylligands normally react in 1 to 4 hours at ambient temperatures there appears no significant advantage in using higher temperatures.
• the presence of sodium chloride in the reaction slurry typically at 0.25 to 0.5 Molar is found to facilitate dye incorporation by a common ion effect. Generally the presence of sodium chloride approximately doubles the dye binding although excessive amounts may reverse this effect.
• chloride groups in the solid medium may have an adverse effect on protein-binding and hence when dichloroligands are used the above process should preferably be followed by a further step to convert any free chloride groups to a less harmful groups such as amine groups.
• protein-binding solid media comprising a protein-binding ligand containing triazinyl groups bound directly to a matrix and having substantially the structure
• R 1 ' and R 2 are as defined above said ligand being present in a proportion of at least 7.5 mg, preferably at least 15 mg per grm of dry matrix.
• the matrix is preferably an agarose polymer.
• the protein-binding solid media may be used in the conventional manner for the separation and purification of proteins by affinity chromatography.
• a crude or semi-purified biological material normally in a buffered solution at a concentration of 1 to 20, but exceptionally up to 100, mg protein per ml, is contacted, normally in a column,with the solid medium.
• the retained protein may be eluted by standard techniques such as by using a solution of different pH, buffer composition or ionic strength, or containing a co-substrate, cc-factor, inhibitor,
• the media of the present invention are capable of performing simple and highly specific purifications of a wide range of proteins, especially blood proteins and enzymes, both animal and bacterial.
• selected media may be used to purify albueinkij-uases such as glycerokinase and especially urokinase, nucleases including restriction endonucleases, dehydrogenases such as glyceraldehyde-3-phosphate dehydrogenase or ⁇ -hydroxy-butyrate dehydrogenase, esterases such as cholinesterase, or DNA or RNA binding proteins such as DNA lygase.
• albueinkij-uases such as glycerokinase and especially urokinase
• nucleases including restriction endonucleases dehydrogenases such as glyceraldehyde-3-phosphate dehydrogenase or ⁇ -hydroxy-butyrate dehydrogenase
• dehydrogenases such as
• ligands used in these examples were commercial dyes having the following typical structures and Colour Index Constitution Numbers (CTCN).
• CTCN Colour Index Constitution Numbers
• M may sometimes represent a hydrogen atom, but normally represents an alkali metal, usually sodium, ion. All dyes were ether washed before use.
• Ligand B bound to the matrix of Example 9 using the process described by Baird et al, Febs Letters November 1976 pages 61-66 using sodium carbonate as alkali at final contentrations of 0.002 to 0.2N.
• the standing times and analysis procedure were as in Example 9. Results are shown in Table 2.
• Ligands E and H were attached to either Sepharose 4B or Cellulose by the methods of the present invention or Baird et al (supra) at the optimum final alkali concentration for each system. The results are shown in Table 3.
• Example 1 was repeated using varying amounts of water and 10mg/ml ligand solution in order to vary the ligand concentration.
• the final alkali concentration was 0.025N and the incubation period was reduced from 19 to 17 hours.
• the results are shown in Table 4.
• Example 1 was repeated using a monochloro-triazine ligand, ligand A, and a dichloro-triazine ligand, ligand D, at a final alkali concentration of 0.025N. Samples vere withdrawn at various intervals after alkali addition and analysed for ligand content. Results are shown in Table 5.
• Example 2 The procedure of Example 1 was used to bind Ligand E to 1 grm samples of nylon sheet (porosity 30 microns) in the presence of various final concentrations of either sodium hydroxide or sodium carbonate. The amounts of ligand bound were estimated from the residual content of the reaction liquid since the acetic acid digestion procedure cannot be applied to nylon. Results are shown in Table 6.
• Example 1 The process of Example 1 was repeated using ligand B and various matrices at a final sodium hydroxide concentration of 0.025N and mixing for 74 hours. The weights of ligand bound are shown in table 7.
• ligands E, H & I were bound to Sepharose 4B by the methods of the present invention
• the method of the present invention gives higher bound ligand concentrations than either of the prior art techniques without cyanogen bromide activation and gives ligand binding comparable to that obtainable with cyanogen bromide activation.
• the stabilities of the columns of ligands E and I bound to the matrix by different methods were compared by preparing 2ml columns in 10mM- potassium phosphate (pH 7.5) and storing these at ambient temperature (18-22oC). The columns were periodically viashed with 20-35ml of the same buffer and the ligand content of the washings estimated by spectrophotometry. The percentage of ligand remaining on the column was estimated.
• lines a and b show the leaching pattern for columns of ligands E and I respectively bound by the method of the present invention
• lines c and d shows the equivalent patterns for columns of ligands E and I.bound by the method of US Patent 4,016,149 using cyanogen bromide activation.
• glycarokinase derived from Bacillus stearothermophilus and having an activity of 25 Units per Eg protein (U/mg) was dissolved at a concentration of 5 to 15 mg/ml in 10mM potassium phosphate (KP) buffer at pH 7.5 and run onto a column of the solid medium. The column was then washed with buffer and the bound enzyme eluted with a solution of 5mM MgCl 2 and 5mMATP in the same buffer. The amounts of enzyme bound by the various media and recovered are shown in Table 8.
• the product from Example 40 had a specific activity of 120 U/mg and showed a single band on SDS polyacrylamide gel electrophoresis (PAGE) at 58,000 daltons.
• Example 43 was repeated using media comprising various concentrations of ligand E on Sepharose 4B and eluting vith 1M KCl/ 2mM MgCl 2 /2mM ATP in the same buffer. Results are shown in Table .
• EXAMPLE 53
• Example 47 was repeated using a medium comprising 5.6mg ligand E per grin of Sepharose kB with cyanogen bromide activation. Results are shown in Table 9. TABLE 9
• EXAMPLES 54 and 55 The partially purified enzyme as used in Examples 43 to 46 was dissolved in 10mM KP buffer pH 5.5 and run onto columns comprising ligand F on Sepharose 4B at 0.2mg/g and 0.7mg/g respectively. The amounts bound were 100 and 200 units (not saturated). V/hen washed with 50mM KP at pH7.5 virtually all the bound enzyme was eluted from the 0.2mg/g column but none from the 0.7mg/g column. All the enzyme could have been eluted from either column with 1M potassium chloride.
• EXAMPLE 56 The partially purified enzyme as used in Examples 43 to 46 was dissolved in 10mM KP buffer pH 5.5 and run onto columns comprising ligand F on Sepharose 4B at 0.2mg/g and 0.7mg/g respectively. The amounts bound were 100 and 200 units (not saturated). V/hen washed with 50mM KP at pH7.5 virtually all the bound enzyme was eluted from the 0.2mg/g column but none
• a partially purified cell extract from Pseudomonas bacteria containing 24 units/mg protein of carboxy-peptidase G was dissolved at about 10mg/ml in 25mM tris-acetate buffer pH7.0 and run onto a column comprising ligand G on Sepharose 4B at 0.39mg/g.
• the column was washed with the same buffer and the enzyme eluted with 2mM para aminobenzoyl glutamate (PABA-G).
• the product had a specific activity of 150 Units/mg. The elution could also be performed with 1M KCl.
• Example 56 was repeated using columns comprising ligand B on Sepharose 4B at 0.1mg/g and 0.65 mg/g respectively.
• the enzyme could not be eluted from the 0.65 mg/g column with 2mM PABA-G, but could be eluted from either column with 2M KCl.
• EXAMPLE 59
• a crude cell extract from 1Kg of cells of Rhodopseudomonas spheroides containing about 0.1 U/mg protein of ⁇ -hydroxy-butarate dehydrogenase was suspended at 30-40 mg protein/ml in 10 M KP buffer pH 7.5. This was run onto a 2 litre column comprising ligand A on Sepharose 4B at 0.44 mg/g. The column was washed with the buffer and eluted with IMKCl in 10 mM KP pH.7.5. Recovery was greater than 95% with a specific activity of 3-4 U/mg.
• Example 56 was repeated using the media produced in Examples 39-42 respectively.
• the amounts of enzyme bound are shown in Table 10. Recoveries on elution with 1HKCl were 85-90%. in all cases
• Centrifuged equine ⁇ erun was Malawially purified by adsorption on DEAE cellulose to yield a protein preparation containing 0.08U cholinesterase/mg protein. This was frecse-dried and then dissolved at 20mg protein/ml in 10mM KP buffer at pH6.8 and run onto a column comprising ligand C on Sepharose 4B at 0.5mg/g. 14 U/ml of packed column were bound. The column was washed with buffer and eluted with the same buffer containing 1MKC1 to give 80% yield of a protein preparation having a specific cholinesterase activity of 4U/mg protein.
• EXAMPLE 65 Crude human urine was concentrated about 20 to 40 times by ultrafiltration using a hollow fibre unit (Trade Mark "Ainicon” type HIDP10). The concentrate (containing 2.5mg protein/ml) was run onto a column comprising ligand F on Sepharose 4B, at 0.6mg/g, preequilibrated with 10mM KP buffer pH7.5. The column was washed with buffer and eluted with 1M KCl pH7.5 to yield a urokinase preparation having a specific activity 200-200 times that of the concentrate.
• Example 65 was repeated using ligands B and E respectively. Satisfactory separations were obtained in both cases.

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• 1979
  • 1979-01-22 JP JP54500355A patent/JPS635133B2/ja not_active Expired
  • 1979-01-22 WO PCT/GB1979/000016 patent/WO1979000541A1/en unknown
  • 1979-01-22 DE DE7979900153T patent/DE2966596D1/de not_active Expired
  • 1979-08-13 EP EP79900153A patent/EP0007917B1/en not_active Expired
• 1983
  • 1983-10-26 US US06/545,546 patent/US4546161A/en not_active Expired - Fee Related

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